The invention relates to novel recombinant glycoproteins that act as messenger substances, signaling substances, promoters, stimulators and initiators in a multitude of ways in th animal, especially human, circulation system; to processes for their preparation, to pharmaceutical compositions containing them and to their uses. The invention in particular relates to new recombinant human glycoproteins (rh glycoproteins), preferably novel rh differentiation factors, especially rh erythropoietin; to novel rh growth factors, especially rh CSF (colony-stimulating-factor), rh GMCSF (granulocytes-monocytes-stimulating factor); to novel rh thrombolytic agents, especially rh tPA (tissue-plasminogen-activator) and rh urokinase; to novel rh thromboprophylactic agents, especially rh antithrombin III; to novel rh coagulation factors, especially rh factor VIII and rh factor IX; to novel rh interferons, especially rh xcex1-, xcex2- and xcex3-interferons; and to novel rh interleukins, especially rh IL-2, IL-15, IL-16 and IL-17; to processes for their preparation; to novel pharmaceutical compositions containing them and to the uses thereof.
Glycoproteins are proteins containing covalently-bound carbohydrates (sugars). Glycoproteins, especially human glycoproteins, act as messenger substances, signaling substances, promoters, stimulators and initiators in metabolic processes of animals and human beings, which directly or indirectly influence these metabolic processes. Usually they are formed in vivo in the body or biosynthetically and, after having fulfilled their specific function, they are again excreted thereof or broken down therein. The glycoproteins having been formed in a natural way within the body (so called xe2x80x9cnative glycoproteinsxe2x80x9d) are characterized in that they are substituted at the amine group of the aminoglycan radical by an acetyl group. These native glycoproteins formed in a natural manner in vivo or by biosynthesis (see German patent 4 311 580) in human and animal are effective means for stimulating the growth and the differenciation of human and animal cells of the immune system and for inhibiting the adhesion of leucocytes, thrombocytes and tumor cells at the endothelial cells of blood vessels; for stimulating the immune system, especially the T-lymphocytes, for defense against infection, for treating immune deficiencies, tumor diseases including metastasing processes, infectious diseases and circulatory collapse, especially occlusions of vessels and septicemia; for inhibiting the bonding of a ligand at its sialylated cell surface receptor to a host cell; for inhibiting the bonding of a microphage or a toxin to the host cell via a sialylated receptor by an in vivo-modulation of neuraminic acids; for the biosynthetic preparation of ligands or receptors having a modified neuraminic acid radical and for the use thereof for the competition of physiological or pathological ligand-receptor-interactios; for influencing in vitro the course of infection by human immunodeficiency viruses as well as the in vivo-prevention of an infection by human immunodeficiency viruses; and for the treatment of parasitic diseases.
The glycoproteins occuring in animal organisms are important components of cell membranes as well as soluble components of body fluids and of the extracellular matrix. The carbohydrates are linked to form chains (oligosaccharides) and they can be linked to the protein backbone in different ways. As important components of the cell membrane they contain sialic acid (derivatives of 2-keto-3-deoxy-D-glycero-D-galacto-nonulopyranosidonic acid (KDN)), which plays an important role in biological processes.
The oligosaccharides of glycoproteins are classified according to their mode of linkage to the protein. The oligosaccharide of thioacidglycoproteins are mostly bound N-glycosidically to an asparagine radical of the polypeptide chain (N-glycans). This group comprises dissected glycoproteins having different functions, e.g. soluble enzymes, immunoglobulins and hormones on the one hand, and membrane glycoproteins, e.g. membrane enzymes, transport proteins as well as receptor proteins on the other hand. A further group are the oligosaccharides which are bound O-glycosidically via a galatose, N-acetyl-galactosamine or xylose radical to a serine- or threonine radical of the polypeptide chain (O-glycans). They occur together with N-glycans also in immunoglobulins and other glycoproteins. The O-glycans also comprise the oligosaccharides of proteoglycans which are characterized by a particularly high proportion of carbohydrates. In these glycoconjugates occuring in the extracellular matrix the oligosaccharides can be bound via a galactose, N-acetylgalactosamine or xylose radical to a polypeptide backbone.
The glycoproteins consisting of monosaccharides and protein often are summarized with the glycolipids as glycoconjugates. The sugar component of the glycoproteins comprising, with a few exceptions, less than 50% of the total glycoproteins are linked via splittable O- or N-glycosidic bonds to the peptide part. As carbohydrates in the glycoproteins hexoses (galactose, mannose, more seldom glucose), N-acetylhexosamines, N-acetylneuraminic acid, fucose and others can be found. For identification and determination of the glycoproteins mainly affinity chromatography using plant lectins as ligands (e.g. concanavalin A, wheat germ agglutinins and others) is suited.
Almost all membrane glycoproteins, serum proteins, plasma proteins, and blood group substances are glycoproteins as are many enzymes and proteohormones, all antibodies, the chalones, mucins, lectins, bindins, fibronectins, the intrinsic factor and similar proteins. As membrane or cell surface proteins some glycoproteins play an important role in the pathogenicity of viruses. In this case, as in other receptor-specific cellular interactions, the carbohydrate components are responsible for the recognition process at the molecular level. Some bacteria and viruses adhere to their target cells via specific sugar structures on receptors.
Oligosaccharide structures are particularly important also in view of cell-cell and cell-matrix interactions. Thus, the oligosaccharides of glycoproteins mediate the adhesion of neuronal cells and the bonding of lymphocytes to specific endothelial cells. Furthermore, oligosaccharides can serve as the antigenic determinants of glycoproteins. Also during embryogenesis and organogenesis, carbohydrate-carbohydrate interactions are essential to specific cell recognition. The malignant transformation of cells is accompanied by characteristic changes in the oligosaccharide structures of glycoproteins. The extent to which altered oligosaccharide structures of membrane and intercellular glycoproteins of tumor cells are the cause or result of tumorigenesis and metastasis is not known until yet.
For the treatment of diseases related to the immune system, substances have to be administered that reinforce the immune response by stimulating the cells of the immune system. The search for active substances that stimulate the immune system is therefore a preferred goal of pharmacological research. Active immunstimulants having minimal side effects, however, are not known until yet.
Therefore, the object of the invention was to find new substances by means of which it is possible to influence the metabolic processes controlled by glycoproteins as messenger substances, signaling substances, promoters, stimulators and initiators, more effectively, more specifically and more selectively than until today. It was in particular the object of the invention to find such novel glycoproteins acting as messenger substances, signaling substances, promoters, stimulators and initiators in human and animal metabolism and which can be administered in lower doses and therefore have lower side effects than the corresponding native substances.
Now it has been found that this object can be achieved according to the present invention by novel glycoproteins which are different from the abovementioned native glycoproteins in that the naturally occurring acetyl substitution in the aminoglycan radical ist repaced by other acyl groups, in particular by a (C3-C7)-acyl group or a hydrocarbyl group, especially a (C3-C7)-alkyl, -alkenyl or -alkinyl group. In fact, it has been shown that the thus obtained novel compounds exhibit an altered, in particular a longer, pharmacokinetics, compared with the biological half-life time of the corresponding, already known and commercially available native glycoproteins having an N-acetyl group in their aminoglycan radical. These novel glycoproteins can be prepared in a manner as described below, in particular by genetic engineering where recombinant glycoproteins, especially recombinant human glycoproteins (rh glycoproteins) are obtained.
Due to their altered, especially longer, pharmacokinetics these substances can be used in substantially lower doses which result in substantially reduced undesired side effects compared with those found by using the corresponding native substances. This applies in particular when the novel glycoproteins are used in a form which is mono- or poly-acylated, preferably -acetylated, at the OH groups of the monosaccharide part.
Subject-matter of the present invention are according to a first aspect novel recombinante glycoproteins, especially rekombinant human glycoproteins (rh slycoproteins) having the general formula (I) 
wherein:
Rxe2x80x21 is a linear or cyclic, unbranched or branched, optionally mono- or poly-hydroxylated and/or -ketylated (C3-C7)-acyl radical, in particular (C3-C7)-alkanoyl radical, or a linear or cyclic, unbranched or branched, optionally mono- or polyhydroxylated and/or -ketylated (C3-C7)-hydrocarbyl radical, in particular (C3-C7)-alkyl, -alkenyl or -alkinyl radical;
R2, R3, R4 and R5, which can be the same or different, each are hydrogen, a linear or branched alkyl radical having 1 to 20 carbon atoms (CnH2n+2; n=1 to 20); a linear or branched alkenyl radical having 3 to 20 carbon atoms (CnH2n; n=3 to 20, the position of the double bond at Cn is at n=2 to 19); a linear or branched alkinyl radical having 3 to 20 carbon atoms CnH2n-2, n=3 to 20, the position of the triple bond at Cn is at n=2 to 19); an alkenyl or alkinyl radical having 2 or more double bonds and triple bonds, respectively, and having 4 to 20 carbon atoms; an aryl radical having 6 to 20 carbon atoms; a linear or branched, saturated or mono- or poly-unsaturated acyl radical (xe2x80x94COxe2x80x94R1) having in total 1 to 20 carbon atoms, including the mono- or poly-hydroxylated analogues thereof; an aroyl radical having 6 to 20 carbon atoms; a carbonylamide radical of formula xe2x80x94CONH2 or xe2x80x94CONHR1; a linear or branched, saturated or mono- or poly-unsaturated thioacyl radical (xe2x80x94CSxe2x80x94R1) having in total 1 to 20 carbon atoms; or a thiocarbamide radical of formula xe2x80x94CSxe2x80x94NH2 or xe2x80x94CSxe2x80x94NHR1;
where R1 each represents hydrogen, a linear or branched, saturated or mono- or poly-unsaturated alkyl radical having 1 to 20 carbon atoms and where each of the above-mentioned radicals with the exception of H optionally can be mono- or poly-substituted by halogen, hydroxy, epoxy, amine, mercaptan, phenyl, phenol or benzyl groups and
T is a mono- or di- or oligo-saccharide radical having up to 40 glycosidically linked, optionally branched sugar radicals, the saccharide radical containing furanose and/or pyranose rings and containing 5 to 230 carbon atoms and being N- or O-glycosidically bound to polypeptides.
The preferred meanings in the above general formula (I) are as follows:
R2, R3, R4 and R5, which can be the same or different, each represent hydrogen, a linear or branched alkyl radical having 1 to 7 carbon atoms; a linear or branched alkenyl radical having 3 to 10 carbon atoms; a linear or branched alkinyl radical having 3 to 10 carbon atoms; an alkenyl or alkinyl radical having 2 or more double bonds and triple bonds, respectively, and having 7 to 12 carbon atoms; a phenyl radical; a linear or branched, saturated or mono- or polyunsaturated acyl radical (xe2x80x94COxe2x80x94R1) having in total 1 to 7 carbon atoms, in particular an acetyl radical, including mono- or poly-hydroxylated analogues thereof; an aroyl radical having 6 to 10 carbon atoms; a carbonylamide radical of formula xe2x80x94CONH2 or xe2x80x94CONHR1; a linear or branched, saturated or mono- or poly-unsaturated thioacyl radical (xe2x80x94CSxe2x80x94R1) having in total 1 to 7 carbon atoms; or a thiocarbamide radical of formula xe2x80x94CSxe2x80x94NH2 or xe2x80x94CSxe2x80x94NHR1;
where R1 each represents hydrogen or a linear or branched, saturated or mono- or poly-unsaturated alkyl radical having 1 to 20 carbon atoms and where each of the above-mentioned radicals with the exception of H optionally can be mono- or poly-substituted by fluorine, chlorine, bromine or iodine, hydroxy, epoxy, amine, mercaptan, phenyl, phenol or benzyl groups.